Isolation of Mouse Embryonic Fibroblasts (MEFs): Difference between revisions

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From Zolov et al (under review)

==Materials==

* Collagenase, type II (LS004204, Worthington, US)

* Clean surgical blades

* DMEM/PSG/10% FBS

==Generation of Mouse Primary Fibroblast Cells==

~~Fibroblasts~~ were prepared from tails and legs of P0 pups. ~~Tissues from individual animals were kept separate, disaggregated with~~ a surgical blade ~~and digested with 500 U/ml type II collagenase (LS004204, Worthington~~, ~~US) in 5 ml RPMI media (Cat~~. # ~~1640, Invitrogen, US) supplemented with~~ 10% ~~FBS, 2 mM L-glutamine and penicillin~~/~~streptomycin~~ for 12 hours at 37°C. After incubation, cells were harvested at 200 g for 5 min ~~and plated~~ in 100 mm dishes~~, in fresh RPMI medium supplemented with 10% FBS, 2 mM L-glutamine~~ and ~~penicillin/streptomycin. Experiments were performed on cells that were passaged 1-3 times.~~

# MEFs were prepared from tails and legs of P0 pups.

# Disect out all four limbs plus the tail. Place one small piece into PCR tube/plate for genotyping

# Using a clean surgical blade, chop into tiny pieces.

# Place into 5 mL of collagenase in DMEM/PSG/10% Serum (500U/mL) in a 15 mL falcon tube

# Incubate in incubator for 12 hours at 37°C.

# After incubation, cells were harvested at 200 g for 5 min

# Wash cells once with warm media, then resuspend in DMEM.

# Plate into 2-3 100 mm dishes and change media afer 1d

# Split when confluent

@@ Line 1: / Line 1: @@
 From Zolov et al (under review)
+==Materials==
+* Collagenase, type II (LS004204, Worthington, US)
+* Clean surgical blades
+* DMEM/PSG/10% FBS
 ==Generation of Mouse Primary Fibroblast Cells==
-Fibroblasts were prepared from tails and legs of P0 pups. Tissues from individual animals were kept separate, disaggregated with a surgical blade and digested with 500 U/ml type II collagenase (LS004204, Worthington, US) in 5 ml RPMI media (Cat. # 1640, Invitrogen, US) supplemented with 10% FBS, 2 mM L-glutamine and penicillin/streptomycin for 12 hours at 37°C. After incubation, cells were harvested at 200 g for 5 min and plated in 100 mm dishes, in fresh RPMI medium supplemented with 10% FBS, 2 mM L-glutamine and penicillin/streptomycin. Experiments were performed on cells that were passaged 1-3 times.
+# MEFs were prepared from tails and legs of P0 pups.
+# Disect  out all four limbs plus the tail.  Place one small piece into PCR tube/plate for genotyping
+# Using a clean surgical blade, chop into tiny pieces.
+# Place into 5 mL of collagenase in DMEM/PSG/10% Serum (500U/mL) in a 15 mL falcon tube
+# Incubate in incubator for 12 hours at 37°C.
+# After incubation, cells were harvested at 200 g for 5 min
+# Wash cells once with warm media, then resuspend in DMEM.
+# Plate into 2-3 100 mm dishes and change media afer 1d
+# Split when confluent